Tumor metastasis is a multistep process requiring detachment of malignant cells from the primary tumor, penetration of blood or lymph vessels and attachment to endothelium of distant organs and formation of new tumors. The ability of disseminating cancer cells to establish metastases in secondary organs is regulated by a combination of factors, including access to the organ microvasculature and specific host-tumor interactions [Radinsky and Fidler, 1992, In Vivo 6:325-331]. Hematogenous dissemination brings cancer cells into contact with leukocytes, platelets, and endothelium [Nicolson, 1989, Curr. Opin. Cell. Biol. 1:1009-1019, Bevilaqua et al., 1989, Science 243:1160-1165]. The attachment of circulating tumor cells to the vascular endothelium of the target organ is thought to be a key step in the metastatic cascade. Once tumor cells adhere to endothelial cells (EC), they penetrate through the EC layer, moving into subendothelial tissues where metastasis is established.
Studies of leukocyte transmigration have suggested that the specificity of the interaction between circulating lymphocytes and the microvascular endothelium may be determined by the outcome of a series of sequential adhesion molecule-ligand interactions involving complex carbohydrate structures on the surface of leukocytes and adhesion molecules on the surface of endothelial cells. It appears likely that the process of tumor extravasation is mediated through a series of analogous adhesive interactions [Brodt, 1996, In: Cell adhesion and invasion in cancer metastasis, vol. 21 pp. 167-242, Brodt, ed., Landes, Austin and Springer-Verlag, Berlin].
Prominent among the vascular endothelial cell adhesion molecules implicated in both leukocyte and tumor cell transmigration are members of the selectin family. This family of adhesion molecules supports the adhesion of leukocytes to the vessel wall through the recognition of specific carbohydrate structures and thereby mediates critical cell-cell interactions in processes such as leukocyte trafficking, thrombosis, acute and chronic inflammation and ischemia reperfusion injury [Bucher and Picker, 1996, Science 272:60-66; Lasky, 1995, Annu. Rev. Biochem. 64:113-139; Varki, 1994, Proc. Natl. Acad. Sci. USA 91:7390-7397; Bevilaqua and Nelson, 1993, J. Clin. Invest. 91:379-387]. Selectins recognize and bind specific carbohydrate antigens expressed on tumor cell surfaces and mediate the initial interaction between tumor cells and endothelium [Mannori et al., 1995, Cancer. Res. 55:4425-4431 (Mannori I)].
Selectins are subdivided into E-, L-, and P-selectin subgroups. Inflammatory mediators such as tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) induce vascular endothelium to express E- and P-selectin. E-selectin is a calcium-dependent molecule expressed by activated vascular endothelium during the process of leucocyte recruitment. E-selectin binds to glycoconjugates carrying a terminal tetrasaccharide Lewis (Le) antigen, sialyl-LeX (SA-LeX), [NeuAcα2,3Galα1β1,4(Fucα1,3) GlcNAcβ1,3Ga1β1,4Glcβ1-R], but displays higher affinity for the SA-Lea structure ([NeuAcα2,3Ga1β1,3(Fucα1,4) GlcNAcβ1,3Galβ1,4Glcβ1-R], a positional isomer of SA-LeX.
P-selectin is expressed by activated platelets and endothelial cells. P-selectin has been shown to mediate adhesive interactions of some colon adenocarcinoma cells with thrombin-activated platelets [Mannori I, cited above]. During metastatic dissemination, tumor-platelet adhesion may result in the formation of neoplastic emboli that facilitate the arrest of tumor cells in the microvasculature of organs [Karpatkin and Pearlstein, 1981, Ann. Intern. Med. 95: 636-641].
L-selectin is constitutively expressed by a majority of leukocytes, including neutrophils, monocytes, natural killer cells and most lymphocytes. L-selectin expressed on leukocytes can support interaction with cancer cells, enhancing metastatic potential [Welch et al., 1989, Proc. Natl. Acad. Sci. USA 86:5859-5863]. Other studies demonstrate that the engagement of L-selectin on lymphocytes can stimulate their anti-tumor cell cytotoxic activity [Seth et al., 1991, Proc. Natl. Acad. Sci. USA 88: 7877-7881].
Selectin recognition of carbohydrate ligands involves primarily the N-terminal of C-type lectin domain, influenced by the EGF-like domain, and to a lesser degree the short consensus repeats [Kolbinger et al., 1988, Biochemistry 35:6385-6392; Revelle et al., 1996, J. Biol. Chem. 271:16160-16170; Li et al., 1994, J. Biol. Chem. 269:4431-4437]. Mutagenesis studies of the lectin domain of the selectins [Erbe et al., 1993, J. Cell. Biol. 120:1227-1235] and the crystal structure of the E-selectin lectin plus EGF-like domain solved at 2.0 Å resolution [Graves et al., 1994, Nature 367:532-538] have identified a number of positively charged residues involved in the binding to the SA-LeX carbohydrate ligand. Most of these residues are identical in all selectins and they are likely to recognize essential carbohydrate moieties such as sialic acid and fucose of SA-LeX. The identified residues are derived from noncontiguous sequences at both the N- and C-terminus of the lectin domain.
Although adhesion pathways utilized by different tumors exhibit considerable diversity, metastasis apparently involves the interaction of at least one member of the selectin family of adhesion molecules with the antigens SA-Lea and/or SA-LeX. These ligands may be involved in tumor metastasis by mediating binding of blood-borne tumor cells via E- and/or P-selectin to vascular endothelium [Giavazzi et al., 1993, J. Clin. Invest. 92:3038-3044; Dejana et al., 1992, Lab. Invest. 66:324-3130, Takada et al., 1993, Cancer. Res. 53:354-361; Sawada et al., 1994, J. Biol. Chem. 269:1425-1431]. Cancer cells that express both SA-Lea and SA-LeX undergo SA-Lea-mediated adhesion almost exclusively, possibly due to the higher affinity for the SA-Lea structure, or differential presentation of this oligosaccharide determinant. Thus, SA-Lea might play a major role as a ligand in the E-selectin dependent adhesion to EC in vivo. Indeed, SA-Lea specific monoclonal antibodies (MAbs) were inhibitory for adhesion of colon carcinoma cells to human umbilical cord vein endothelial cells (HUVEC).
In vivo studies have provided firther evidence of the potential importance of the carbohydrate ligand/E-selectin interaction in tumor metastasis [Brodt et al., 1997, Int. J. Cancer 71:612-619; Mannori et al., 1997, Am. J. Pathol. 151:233-243 (Mannori II), Biancone et al., 1996, J. Exp. Med. 183:581-587].
Alternatively, some carcinoma cells do not express these carbohydrate determinants (i.e., SA-LeX and SA-Lea) and yet they can attach to EC prior to activation. Further, this adhesion is not augmented by cytokine treatment, suggesting E-selectin-independent adhesion [Iwai et al., 1993, Int. J. Cancer 54:972-977, Tozeren et al., 1995, Int. J. Cancer 60:426-431; Miyake et al., 1992, New Eng. J. Med. 327:14-18; Garrigues et al., 1992, J. Cell. Biol. 125:129-142].
Studies have also demonstrated the role of oligosaccharides in inflammatory responses. Neutrophil extravasation is enabled by a multistep process initiated by the selectin family [Kansas, 1996, Blood 88: 3259-3287]. Neutrophil-endothelial cell interaction mediated via the selectins in the context of vascular shear flow, are characterized by transient tethering of the neutrophils, followed by rolling of the neutrophil along the endothelial surface of the vessel wall. Studies in vivo and in vitro indicate that selectin-dependent neutrophil rolling is essential to subsequent events in the transmigration process. Neutrophils are exposed to endothelial cell derived IL-8, platelet-activating factor and other neutrophil-activating molecules [Lowe, 1997, In: The selectins: Inhibitors of leukocyte endothelial adhesion, pp. 143-177, Vestweber, ed., Harwood Academic Publishers, Reading, UK], which in turn promote activation of neutrophil P2 integrins, leading to integrin-dependent firm adhesion to the integrin receptor ICAM-1, and finally to neutrophil extravasation, possibly via homophilic interaction of platelet/endothelial cell adhesion molecule 1.
The expression of ligands for selectins, particularly E-selectin, by both neutrophils and carcinoma cells raises the possibility that metastases are equivalent to the inflammatory process in which tumor cells, particularly carcinoma cells, use the same molecular mechanism(s) for cancer cell-EC interaction as lymphocytes, through the adhesion interaction of the endothelial cell selectins with the tumor-associated carbohydrate ligands, e.g., SA-LeX, SA-Lea, and LeY.
In addition to their role in cell adhesion, carbohydrate structures also play a role in angiogenesis. Aberrant angiogenesis can occur in a variety of pathologic conditions. Neovascularization of tumors occurs by aberrant stimulation of normally quiescent endothelial cells to migrate, proliferate and form new capillary blood vessels [Ingber and Folkman, 1989, J. Cell. Biol. 109:3317-3330]. The experimental evidence suggests that E-selectin and its ligand SA-LeX function in angiogenesis [Nguyen et al., 1982, J. Biol. Chem. 267:26157-26165]. Thus, proliferating microvascular endothelium presents a potential target for anti-cancer and anti-angiogenic therapies through the inhibition of E-selectin-dependent carbohydrate-mediated interactions [Folkman, 1995, N. Engl. J. Med. 333:1757-1763].
Although recent studies suggest the importance of carbohydrate ligand-cell adhesion interactions in tumor metastasis, angiogenesis and inflammatory responses, the complex nature of the carbohydrate ligands involved has long hampered studies of these processes. The difficult chemical or enzymatic synthesis required by these complex carbohydrate ligands and the technical complexity involved in analyzing the functional/structural interactions of these ligands with selectins at the molecular level have severely hindered the development of anti-adhesion therapeutics for treatment of these disease processes for which there is no effective treatment.
Many peptide mimics of carbohydrate structures have been described in the literature [see, e.g., Agadjanyan, M. et al, 1997, Nature Biotechnol. 15: 547-551, among others] including those binding with high affinity to E-selectin [Tsukida, T. et al, 1998, J. Med. Chem. 41: 4279-4287]. A peptide that mimics the GD1 ganglioside, also involved in cell adhesion and metastasis of melanoma cells, has been recently described [Ishikawa, D. et al, 1998, FEBS Lett. 441: 20-24]. This peptide isolated from a peptide phage display library using an anti-GD1 antibody inhibits metastasis in an in vivo model.
Thus, there remains a long-felt and acute need for the development of techniques and probes for the study of complex carbohydrate ligand-cell adhesion molecule interactions, and for the development of anti-tumor, anti-inflammatory and angiogenesis-blocking therapeutics based on the selective inhibition of these interactions.